The practical utility of many drugs having potentially useful biological activity is often hindered by problems in delivering such drugs to their targets. The delivery of drugs and other compounds into cells generally occurs from an aqueous cellular environment and entails penetration of a lipophilic cell membrane to gain cell entry.
Oligonucleotides and their analogs are one class of potentially useful drugs whose practical utility has been impeded due to insufficient cellular uptake, and it has been proposed heretofore to enhance uptake of oligonucleotides through conjugation of arginine-rich peptides containing non-α amino acids (see, for example, Chen, Zhang et al. 2003; Abes, Moulton et al. 2006; Youngblood, Hatlevig et al. 2007; and Wu et al. 2007). The use of arginine-rich peptides has been reported for the transport of therapeutic drugs, more generally (see, for example, Rothbard, Kreider et al. 2002).
Studies by the inventors and others (Chen, Zhang et al. 2003; Abes, Moulton et al. 2006; Youngblood, Hatlevig et al. 2007) have established that incorporation of unnatural amino acids can confer enhanced stability to peptide carriers and enhanced antisense activity to conjugated oligomers, and therefore improve the potential of CPPs (cell penetrating peptides) to deliver therapeutic macromolecules.
Parent U.S. application Ser. No. 12/217,040 discloses studies showing that two of the CPPs reported in the application are effective in selectively targeting oligonucleotides to muscle tissue, particularly heart muscle, but also including quadricep (skeletal) muscle. These two peptides have the generic sequence (RXRR(B/X)R)2XB, where R is arginine; B is β-alanine; and each X is —C(O)—(CH2)n—NH—, where n is 4-6. Additional studies reported herein confirm the ability of these two CPPs to enhance the uptake and functioning in muscles of oligonucleotide antisense compounds conjugated to one of the CCPs.
The parent application disclosed and claimed the use of these two CPPs for targeting antisense oligonucleotides to muscle tissue, in treating certain muscle pathologies. For example, in treating Duchenne muscular dystrophy (DMD), an oligonucleotide designed to promote exon skipping in a mutated dystrophin pre-mRNA (for purposes of restoring the proper reading frame in a mutated dystrophin mRNA), is conjugated to one of the CPPs, for enhanced uptake and functioning the oligonucleotide in muscle tissue, including both skeletal and heart muscle. In treating DMD, it is advantageous to effectively target and treat heart muscle, since improvement in skeletal muscle function alone can place a DMD-compromised heart under even greater stress.
The present invention applies this strategy additionally to the treatment of myotonic dystrophy MD1 and MD2 in muscle tissue, including skeletal and heart muscle tissue. This condition is associated with long polyCUG (MD1) and polyCCUG (MD2) repeats in the 3′-UTR regions of the transcript dystrophia myotonica protein kinase (DMPK). While normal individuals have as many as 30 CTG repeats, DM1 patients carry a larger number of repeats ranging from 50 to thousands. The severity of the disease and the age of onset correlates with the number of repeats. Patients with adult onsets show milder symptoms and have less than 100 repeats, juvenile onset DM1 patients carry as many as 500 repeats and congenital cases usually have around a thousand CTG repeats. The expanded transcripts containing CUG repeats form a secondary structure, accumulate in the nucleus in the form of nuclear foci and sequester RNA-binding proteins (RNA-BP). Several RNA-BP have been implicated in the disease, including muscleblind-like (MBNL) proteins and CUG-binding protein (CUGBP). MBNL proteins are homologous to Drosophila muscleblind (Mbl) proteins necessary for photoreceptor and muscle differentiation. MBNL and CUGBP have been identified as antagonistic splicing regulators of transcripts affected in DM1 such as cardiac troponin T (cTNT), insulin receptor (IR) and muscle-specific chloride channel (ClC-1).
MD1 and MD2 are associated with a variety of serious pathologies including muscle abnormalities and weakness, and in the heart, conduction abnormalities.